1
Are the physiological and biochemical characteristics in dandelion plants 1
growing in urban area (Pisa, Italy) indicative of soil pollution? 2
3
G. Vanni, R. Cardelli, F. Marchini, A. Saviozzi, L. Guidi 4
Department of Agriculture, Food and Environment, Via del Borghetto, 80 – 56124 Pisa, 5
Italy 6
Corresponding author: Lucia Guidi, Department of Agriculture, Food and Environment, 7
Via del Borghetto, 80 – 56124 Pisa, Italy; Phone: +39 50 2216613; Email: 8
luciaguidi@unipi.it 9
10
Abstract. Physiological and biochemical characteristics were evaluated in dandelion 11
plants (Taraxacum officinale) naturally growing in an urban environment. The study 12
area was located in the town of Pisa, Italy, and 27 sites were chosen to assess 13
biochemical and physiological features of dandelion plants and the trace metals 14
content in the urban soil. Concentrations of elements including, Cr, Cu, Mn and Zn 15
were analyzed in the soil and shoot and root tissues of dandelion collected from 16
different sites. Chlorophyll a fluorescence analysis, pigment content, antioxidant power 17
and phenols content were measured in dandelion. The results showed a widespread 18
very limited soil pollution of trace metals in the urban sites. However, Zn was uptake 19
and translocated by dandelion even though no damage was observed in plans. On the 20
basis of the obtained results dandelion plant are able to survive in constrained 21
environment thank to the high phenols that are useful to contrast the oxidative stress 22
induced by heavy metals. 23
24
Keywords: Taraxacum officinale, trace element, photosynthetic pigments, antioxidant 25 capacity 26 27 1 Introduction 28
The urban ecosystems are comprised by diverse land uses, including commercial, 29
industrial, residential, transport, recreational, agricultural and natural areas. This 30
determines different habitat for plants, animals and humans. The urban habitat quality 31
comprises the integration of different abiotic and biotic components such as air, soil 32
and water quality, microclimate and the presence of vegetation. Probably, pollution is 33
the most significant characteristic of urban soil (Vrščaj et al. 2008) and this could 34
2
negatively affect the human health. Clearly, trace metals content plays a key role in 35
the pollution (Biasioli et al. 2006). 36
Phytotoxic amounts of trace metals induce in plants an oxidative stress leading 37
to cellular damage (Yadav 2010). In addition, plants accumulate metal ions that disturb 38
cellular ionic homeostasis, the growth usually becomes inhibited and biomass 39
production decreases (Moulis 2010). Trace metals are active in plant metabolic 40
processes, but they can also be stored as inactive compounds in cell walls, so affecting 41
the chemical composition of plants without causing any injury (Nagajyoti et al. 2010). 42
For this reason, vascular plants are frequently used for biomonitoring (Korzeniowska 43
and Panek 2010). However, native plants tolerant to trace metals characterized by a 44
fast growing usually attract more attention as compared with slow growing plants 45
(Massa et al. 2010). For a long time the dandelion (Taraxacum officinale Web.) has 46
been proposed as a good bioindicator for trace elements polluted environment 47
(Savinov et al. 2007; Bini et al. 2012). 48
Mossop et al. (2009) found a positive correlation between Cu and Pb 49
concentrations in soil and in dandelion roots and leaves, whereas for Zn the 50
relationship was found only for leaves. However, these authors considered the sum of 51
the acid-exchangeable, reducible and oxidisable soil fractions of these elements a poor 52
indicator of potential plant uptake. On the other hand, more recently, Gjorgieva et al. 53
(2011), in a study conducted to assess trace metals pollution in Macedonia soils, 54
classified T. officinale as a trace metals accumulator. 55
Dandelion has also a high antioxidant activity due to high content of secondary 56
metabolites (Park et al. 2011; González-Castejón et al. 2012; Davaatseren et al. 2013). 57
Being secondary metabolites important in plant adaptation to environmental stresses, 58
it is presumable that plant species that possess a high content in secondary 59
metabolites can efficiently counteract the environmental stress (Dixon and Paiva 60
1995). 61
Chlorophyll a fluorescence analysis provides a reliable measure of the 62
photosynthetic performance of plants (Maxwell and Johnson, 2000) including trace 63
metals (Küpper et al. 1996; Baumann et al. 2009; Nagajyoti et al. 2010; da Silva et al. 64
2012). Among different parameters achievable from this tool, certainly the Fv/Fm ratio
65
has been extensively used to monitor photoinhibitory damage (Maxwell and Johnson, 66
2000). In the past, the dandelion has already been used for urban pollution monitoring 67
by means of chlorophyll a fluorescence analysis and other photosynthetic parameters, 68
3
although the number of works on the subject is quite low (Lanaras et al. 1994; Sgardelis 69
et al. 1994; Molina-Montenegro et al. 2010). 70
The novelty of this work is represented by the use of T. officinale as bioindicator 71
assessing its healthy status by chlorophyll a fluorescence analysis. This is a first 72
attempt to investigate the soil contamination in the town of Pisa (Italy) in order to 73
establish correlation between trace metal pollution and their relative sources in an 74
urban environment. 75
76
2 Materials and Methods 77
2.1 The study area 78
The study was conducted in the municipal area of the town of Pisa ((latitude 43°25’00N; 79
longitudine 10°43'00E; Italy), an urban environment of approximately 187 km2 and
80
about 90,000 inhabitants. The artificialized surface (impermeable urban area) was 81
approximately 27 km2 (almost 15% of the total area), with a consumption of soil above
82
the average compared with Regional and Provincial data. The built-up area has been 83
steadily increasing, with the largest increases since the 50s (+260% increase from 84
1954 to 2003). Samples were collected at 31 sites around urban areas of the city of 85
Pisa. Sites 1, 2, 8, 9, 10, 11, 12, 16, 25, 26, 28, 29, and 31 are public green areas 86
utilized as playground; Sites 3, 5, 7, 14, 15, 19, 20, 21, 23, 24, 27, and 30 are traffic 87
roundabouts; Sites 4 and 22 are city green squares; Sites 6 and 17 are public school 88
gardens; Sites 13 and 18 are historic public gardens and at a control site (Site 0) 89
nearby the city represented by the rural area of S. Rossore – Migliarino - Massaciuccoli 90
Natural Regional Park (latitude 43°42’48N; longitude 10°21’44E). The control site is a 91
green area, once cultivated and currently naturalized from more than twenty years. 92
Really, the analyses on plants were carried out only in 27 sites because in 4 sites (sites 93
10, 17, 23 and 25) there were no plants that have reached the entire growth cycle. 94
95
2.2 Plant and soil sampling 96
Plants of T. officinale were collected at the flowering stage (from April to May) because 97
in this phase the species was easily identifiable and reached the full development. 98
At each site, three soil samples were randomly collected from the topsoil (depth 99
0-20 cm). Each sample consisted of five sub-samples, each of which was taken within 100
a 2×2 m square, four on the corners of the square and one in the middle. The five soil 101
cores of each sub-sample were mixed to avoid local in-homogeneities. The sampling 102
4
was performed with a stainless steel hand auger and all the soil that was touched by 103
the metallic digging tools were carefully eliminated with a porcelain putty knife, before 104
soil packing in plastic bags, to avoid cross-contamination. In the laboratory, the 105
samples were air-dried at room temperature (20°C), and, after manually removing any 106
plant material, as roots and leaves, they were stored at 4°C until analysis. Most of the 107
main properties of soils, including texture, pH (soil-water ratio 1:2,5), and limestone, 108
were determined according to the standard methods (Sparks, 1996), while organic 109
carbon content was obtained by the difference between total carbon (measured by dry 110
combustion with an automatic C analyzer FKV induction furnace 900 CS, Eltra - F.K.V.) 111
and inorganic carbon (limestone C). 112
113
2.3 Chlorophyll fluorescence measurements 114
The chlorophyll a fluorescence was measured with a PAM-2000 pulse-modulated 115
fluorometer (Heinz Walz, Germany) connected with a leaf clip holder (2030-B Heinz 116
Walz, Germany) as reported in Guidi et al. (2010). The Fv/Fm ratio [(Fm-F0)/Fm] was
117
calculated as an indicator of the maximum quantum efficiency for PSII photochemistry. 118
Estimates of photochemical (qP) and non-photochemical (qN) quenching coefficients
119
were determined as reported by Schreiber et al. (1986) at an actinic illumination at 950 120
(±50) μmol photons m-2s-1. Actual quantum yield for linear electron transport through
121
PSII, ΦPSII was determined as Fm’-Fs/Fm’ where Fs represents the chlorophyll
122
fluorescence yield in steady state conditions (Genty et al., 1989). The maximum 123
electron transport rate was calculated according to Genty et al. (1990) at 950 μmol m
-124
2s-1 PAR using equation: ETR= Φ
PSII × PAR × 0.5 × 0.84 where 0.5 corresponds to the
125
two photosystems and 0.84 is an estimate of the fraction of the absorbed light and PAR 126
is the value for the light intensity given. 127
128
2.4 Photosynthetic pigments content 129
Photosynthetic pigments were determined on three discs of leaf tissue (1.0 cm2 of leaf
130
area) extracted with acetone 80%. Chlorophylls concentrations were calculated from 131
the equations proposed by Porra et al. (1989) whereas carotenoids concentration was 132
calculated from the equation proposed by Lichtenthaler (1987). 133
134
2.5 Methanol extraction of plant tissues 135
5
According to Kang and Saltveit (2002), 1.0 g of sample tissue was homogenised, 136
extracted in 2.5 mL of HPLC methanol and centrifuged at 15,000 g for 20 minutes at 137
4°C. The obtained supernatants were stored at -20°C until used for analysis of the 138
antioxidant capacity, total phenolic compounds and metal chelating capacity. 139
140
2.6 Antioxidant capacity (DPPH) 141
The ability to scavenge DPPH (2,2-diphenyl-1-picrylydrazyl) free radicals was 142
determined according to the method proposed by Brand-Williams et al. (1995) with 143
minor modifications as reported in Landi et al. (2013). Results are expressed as Trolox 144
equivalent antioxidant capacity (TEAC). 145
146
2.7 Total polyphenols (TP) 147
Total polyphenols were determined using the Folin-Ciocalteau method modified by 148
Singleton and Rossi (1965). An aliquot of methanolic extracts was added to distilled 149
water and Folin-Ciocalteau reagent. After 6 minutes, 7% sodium carbonate solution 150
was added and then the mixtures keep at 25°C in the dark for 90 minutes. Total 151
polyphenols content was calculated from a calibration curve, realized using gallic acid 152
as standard. TP content is expressed as mg of gallic acid g-1 fresh weight (FW).
153 154
2.8 Metal chelating capacity (MCC) 155
Metal chelating capacity was determined by the method of Dinis et al. (1994) modified 156
by Du et al. (2009). Briefly, methanolic extracts (1 mL) in 2.8 mL distilled water were 157
mixed with 50 µL of 2 mM FeCl2x4H2O and 150 µL of 5 mM ferrozine
[3-(2-pyridyl)-158
5,6dyphenil-1,2,4triazine-p-p’-disulfonic acid monosodium salt hydrate, 97% 159
(C20H13N4NaO6S2xnH2O)] and the mixtures were thoroughly shaken. After 10 minutes,
160
Fe2+ was monitored by measuring the absorbance of ferrous ion–ferrozine complex at
161
562 nm. The metal chelating capacity was calculated as follows: MCC (%) = [(1-162
Absorbance of sample)/Absorbance of control] x100. 163
164
2.9 Plant trace metals content 165
The content of trace metals was determined on the aerial and root tissues of T. 166
officinale plants, previously separated and dried in an oven at 60°C for 48 h. Briefly, 167
0.5 g samples of plant material were placed in microwave oven (Milestone Ethos 168
labstation; Milestone, Italy) and digested with 65% HNO3 and 30% H2O2. Samples
6
were diluted with distilled water (Baranowska et al. 2002). Digests were analysed for 170
trace metals (Cr, Cu, Mn and Zn) by a flame atomic absorption spectrophotometer 171
(Perkin Elmer Analyst 100, USA). 172
173
2.10 Soil trace metals content 174
The total content of trace metals was determined by the standard method ISO 11466 175
(International Organization for Standardization 1995). Briefly, 1 g of soil sieved to ≈ 80 176
mesh was treated with aqua regia. Pre-digestion was carried at room temperature for 177
16 hours with occasional manual agitation and, later on, digestion was performed for 178
2 hours at 130±2°C. The obtained suspension was then filtered (ashless Whatman 41 179
filter), the filtrate diluted with 0.17 M HNO3 and stored at 4°C until analysis. The
180
determinations of Cr, Cu, Mn and Zn were performed by inductively coupled plasma 181
mass spectrometer (ICP-MS) by Acme Analytical Laboratories Ltd., Vancouver, 182
Canada. 183
184
2.11 Indexes for contamination assessment 185
Geo-accumulation index (Igeo), enrichment factor (EF), and pollution index (PI) were 186
calculated. Igeo was calculated according to the following equation (Müller 1969): 187
Igeo = log2 [Cx/1.5Bx]
188
where Cx is the measured concentration of the metal x and Bx is the 189
geochemical background value of the element. 190
The EF calculation was expressed according to the equation (Lu et al. 2009): 191
EF = [Cx/Cref]sample/[Cx/Cref]background 192
where Cx is the concentration of the metal and Cref was the concentration of 193
reference element for normalization. The reference element utilized for normalization 194
was Fe (Lu et al. 2009). 195
The PI was calculated as the ratio between the metal concentration in the 196
monitored area and the background content (Chen et al. 2005). The bioconcentration 197
factor (BcF) or translocation factor was calculated, too. The BcF is the ratio of the metal 198
concentration of shoots and root portion of dandelion to the metal concentration of the 199
soil (Kim et al. 2003). The translocation ability of metal in dandelion plants was 200
expressed as translocation index (TI) determined as the ratio of metal concentration in 201
the above-ground tissue to its concentration in the root (Salt and Krämer 2000). 202
7 2.12 Statistical analysis
204
All of the performed analyses, were carried out in triplicate and the results were 205
presented as means ± SD. Analysis of variance was performed by ANOVA procedures 206
through CoStat program, 6.311 version (CoHort Software; Monterey, CA, USA). The 207
means were compared using Tukey’s HSD test. Correlation analysis among 208
biochemical, physiological and chemical feature values was performed with NCSS 209
program, 2004 version (Number Cruncher Statistical Systems; Kaysville, UT, USA). 210
211
3 Results and Discussion 212
3.1 Trace metals content in soil 213
The general characteristics of the urban soils of Pisa are reported in Table 1. On the 214
basis of USDA classification, soils of Pisa showed a predominantly sandy-loam texture 215
(76% of the examined soils). Soil pH ranged from 7.2 to 8.3, with a mean of 7.6. On 216
the basis of the USDA terminology, most of soils (68%) were classified as slightly 217
alkaline, 13% were neutral soils while moderately alkaline soils accounted for 19%. 218
The organic C contents of the Pisa urban soils showed a marked variability, with values 219
ranging from 1.32 to 7.57 g C 100 g-1 dry soil and were considerably higher than that
220
of the control (0.90 g organic C 100 g-1 soil).
221
The amounts of Cr, Cu, Mn and Zn in soils of Pisa are summarized in Table 2. 222
The control site showed a content of 39.8 Cr mg kg-1 soil, a value statistically lower
223
than those observed in the remaining areas in which the total amount of Cr varied 224
between 46.4 and 227.5 mg kg-1 of dry soil with a mean values of 71.2 mg kg-1. On the
225
basis of Igeo and EF indexes, approximately 90% of monitored sites showed a small 226
level of Cr pollution, while the remaining soils were classified as moderately polluted. 227
The PI, a more severe index, classified 94% of the examined areas as middle polluted 228
and the remaining as highly polluted. 229
Copper is one of the elements that mostly enrich urban soils. It is assumed that 230
its increase is due to civil and industrial activities, like coal and oil combustion, pesticide 231
and dyes use, but especially to vehicular traffic (US EPA 2006). The total content of 232
Cu ranged widely between 30.94 and 142.35 mg kg-1 whereas in the control site Cu
233
content was 32.24 mg Cu kg-1, a value significantly similar to those found in a small
234
number of soils (about 10%). On average, Pisa urban soils showed Igeo of 0.40, typical 235
of unpolluted areas. On the contrary, the EF value of 1.55 indicated an anthropogenic 236
enrichment, and also PI showed a moderate and widespread pollution. 237
8
The total amount of Mn ranged between 509 and 1063 mg kg-1 while in the
238
control site Mn amount was 526 mg kg-1, a value significantly lower than those
239
observed in the most of monitored areas. Both Igeo and EF classification showed no 240
enrichment or soil pollution by Mn, while PI showed a medium-low pollution level. 241
The main source of Zn enrichment is attributable to the vehicular traffic but the 242
metal may originate also from wearing of brake lining, types and road paved surfaces, 243
losses of oil and cooling liquid, corrosion of galvanized steel safety fence and other 244
road furniture (Blok 2005). The total content of Zn varied remarkably between 61.70 245
and 522.70 mg kg-1. In the control site, Zn content was 51.00 mg kg-1, a value
246
statistically lower than those found in the most of monitored sites. The Igeo index 247
classified most soils as not polluted, the EF indicated a moderate enrichment in 25% 248
of the areas while PI classified 16% of soils as middle polluted and 6% as strong 249
polluted. 250
Despite the pollution level of Pisa urban soils begins to be alarming, at least for 251
some metals, neutral and slightly alkaline soil pH limits the mobility of these elements 252
reducing their availability for plants. 253
254
3.2 Trace metals content in plant tissues 255
Trace metals contents in shoot and root dandelion plants collected in urban sites of 256
Pisa are reported in Table 3. Cr contents of control were 2.7 and 8.7 mg kg-1 for shoot
257
and root portions, respectively. In shoot of plants grown in urban soils Cr varied 258
between 2.0 and 20.0 mg kg-1, while in root it ranged between 4.0 and 14.7 mg kg-1.
259
Values significantly higher than control were recorded in shoot Cr concentration of 260
plants collected in the sites 22 and 24, whereas at radical level the highest Cr 261
concentrations were recorded in the sites 16 and 24. 262
Dandelion tissues of the control site showed Cu values of 18.1 and 19.3 mg kg
-263
1 for shoot and root, respectively. The amounts of the metal did not vary in mostly of
264
the monitored sites, with the exception of site 22 for shoot and sites 7 and 30 for the 265
radical portion in which Cu concentrations were higher than control. 266
As compared to the control site, the level of Mn in shoot was significantly greater 267
only in sites 21 and 22, while in roots Mn was higher than control in sites 7 and 30. 268
In the aerial part, Zn concentration varied between 63.1 and 196.8 mg kg-1, while
269
in the roots it ranged between 40.0 and 155.2 mg kg-1. Zn contents of control plants
9
were 116.7 and 56.7 mg kg-1 for shoot and root, respectively and an increase in root
271
was observed only in the sites 7 and 13. 272
The values of metals concentrations in dandelion tissues grown in sites of urban 273
area of Pisa are similar to those reported by other authors (Finžgar and Leštan 2007; 274
Massa et al. 2010; Ligocki et al. 2011). For the element Zn, dandelion was able to 275
translocate it from roots to the aerial parts; however, for the other elements dandelion 276
showed concentration of the elements similar or lower in shoot as compared to the 277
concentration recorded in root. These results obtained for Zn confirm as dandelion 278
represent an indicator plant (Baker and Brooks 1989). As already found by other 279
authors (Ge et al. 2000), the difference between our results showed little changes of 280
trace element concentrations in tissues of plants grown in urban sites with respect to 281
control. These results are supported by both the bioconcentration factor (BcF) and the 282
translocation index (TI) for Cr, Cu and Zn. In fact, the BcF values relative to both shoots 283
and roots were always below the cut-value of 100, indicating the exclusion of these 284
elements from the dandelion in respect to the soil matrix (Table 4). Also TI pointed out 285
the low translocation of Cr, Cu and Mn from the radical part of the plant, being more 286
concentrated in this plant portion (Table 4). Theonly exception was represented by Zn 287
for which a active translocation from roots to shoot was observed indicating as Towards 288
the shoot, on the other hand, the contents of such heavy metals in dandelion were 289
within the range of non-toxic concentrations (Kabata-Pendias and Pendias 2001). 290
No significant correlations were found between trace metals in soil and in plant 291
tissue with the exception of Mn (Table 5). For this element an increase in root 292
dandelion tissues was found in relation to the increased level in soil. Ge et al. (2002) 293
and Rosselli et al. (2006) reported no correlations between the contents of metals in 294
soil and plant of dandelion grown in polluted soils. The obtained results confirm that 295
dandelion, with respect to Cr, Cu and Mn, is an excluding plant and this is in contrast 296
to what stated by Gjorgieva et al. (2011) who observed that dandelion was a metal 297
accumulators. 298
Zinc was concentrated prevalently in the root portion but, differently to the other 299
elements, its content was above the limits of toxicity (Kabata-Pendias and Pendias 300
2001). In fact, translocation index for Zn was in almost all the sites greater than 1 301
sometimes reaching the value of 2. These values testifying Zn translocation from roots 302
towards the shoot (Table 4). Consequently, the values of BcF of the aerial part, in 303
various sites greater than 100, suggested the partitioning of the metal in aboveground 304
10
portion of the plant (Table 4). Similar results for Zn accumulation in dandelion plants 305
growing in a polluted soil, were reported by Massa et al. (2010). 306
Results indicate that dandelion was an avoider for Cr, Cu and Mn, while, 307
conversely, this species accumulated Zn in shoots even though without evident 308
symptoms of damage such as chlorosis and/or necrosis. 309
310
3.3 Metal chelating capacity 311
Metal chelating capacity (MCC) of T. officinale plants collected from Pisa urban areas 312
is reported in Figure 1. In aerial part, MCC varied in a wide range between 15.6% and 313
57.3% of the control (mean of 31.9%) while in roots it changed between 15.5% and 314
69.7% (mean of 48.9%). MCC of control were 15.2% and 40.3% for root and shoot 315
portion, respectively. Differently from what reported by Seregin and Ivanov (2001), with 316
very few exceptions, MCC did not change in different sites even though differences in 317
MCC were found between shoot and root values. In fact, the latter showed, sometimes, 318
a greater activity (Figure 1). The MCC of the dandelion shoots was not correlated with 319
that of the roots, nor with any other biochemical or physiological parameters (Table 6), 320
while the MCC of roots showed a correlation with the shoot and root phenols content, 321
suggesting a possible involvement of these compounds in the mechanism of trace 322
metals abatement. 323
324
3.4 Chlorophyll a fluorescence and pigment content 325
Maximum quantum yield of PSII (FV/Fm), i.e. potential photosystem II efficiency for
326
photochemistry, in leaves of dandelion grown in monitored site varied in a very narrow 327
range between 0.808 and 0.838 (Figure 2A), value typical of leaves of "healthy" plants 328
(Björkman and Demmig 1987). On the other hand, the actual quantum yield of PSII 329
(PSII; Figure 2B) was meanly 0.602, similar to that of plants grown in control site
330
(0.627). Leaves from site 11 showed the significant highest values of PSII, whereas
331
the lowest values were recorded in sites 22, 24, 26 and 28 (Figure 2B). 332
The photochemical quenching coefficient, qP, i.e. the proportion of open PSII
333
centers, varied in a very narrow range between 0.802 and 0.940 (Figure 2C) and was 334
similar to that recorded in control site. Non-photochemical quenching coefficient (qN),
335
i.e. the excitation energy dissipation through non-photochemical processes, like heat, 336
varied in a range between 0.422 and 0.699 (mean of 0.559) (Figure 2D). Non-337
photochemical quenching coefficient was similar in dandelion leaves from different 338
11
monitored sites, with the only exception of sites 11 and 26. In fact, dandelion leaves 339
from site 11 showed the lowest value of qN and that from site 26 the highest. These
340
results corroborate the values of PSII. In site 11 leaves showed the highest values of
341
PSII and ETR (Figure 2E) and no activation of dissipation mechanisms, i.e. qN, thus
342
no alteration at photosynthetic level was recorded. On the other hand, in the other sites 343
qN increased, reaching the highest level in site 26 characterized by low levels of PSII
344
and ETR (Figures 2 B and E). 345
These results are confirmed also by the significant correlation coefficients 346
obtained from the regression analysis among Chl fluorescence parameters (Table 6). 347
Values of PSII and ETR were significantly correlated with the others but the
348
correlations were negative with qN. The negative correlation between PSII and qN
349
reflects a reduction in the fraction of light energy that is used for photochemistry and 350
an increase in dissipation mechanisms aimed to photoprotection of photosynthetic 351
apparatus (Demmig-Adams and Adams 2006). A significant negative correlation was 352
found between Zn concentration in the soils and Fv/Fm ratio indicating a negative
353
influence of the increase in Zn in the soil and the PSII photochemical quantum yield 354
(Table 5). Even though in experimental conditions, it has been already reported that 355
Zn interacts with the donor side of PSII inhibiting the Hill reaction (Prasad and Strzalka 356
1999) limiting the PSII efficiency (Redondo-Gómez et al. 2011). 357
The total Chl content varied from 0.98 to 2.01 µg cm-2, with a mean of 1.38
358
(Figure 3A), values significantly similar to that found in dandelion leaves grown in 359
control site (1.20 µg cm-2). A significant higher value of total Chl was found in site 28,
360
while the lowest in site 13 (Figure 3A) as compared with the control. As regards 361
carotenoids, their total content ranged between 0.22 and 0.33 µg cm-2, values similar
362
to that of the control site (Figure 3B). No statistical difference among leaves from 363
different sites was detected. Pigment content was not related with the trace metals 364
content in the soil (Table 5) while a strong correlation (0.547) between Chl content and 365
carotenoid was found (Table 6). Conversely, no significant correlation among Chl 366
content and Chl fluorescence parameters, suggesting that these last parameters were 367
not influenced by Chl content. 368
Results from chlorophyll a fluorescence and content underline a low sensitivity 369
of the dandelion photosynthetic mechanism to urban environment pollution. This was 370
already reported by Lanaras et al. (1994) who investigating on the response of T. 371
12
officinale in an urban pollution of Thessaloniki (Greece) found no relation both between 372
Chl amount and the Fv/Fm ratio and pollution levels. In a similar way, more recently,
373
Massa et al. (2010), characterizing trace metals accumulating ability by autochtonous 374
plants grown in an Italian multi-contaminated site, had shown in dandelion plants no 375
significant changes in photochemical PSII efficiency. 376
Although there is evidence that the excess of trace metals inhibits directly the 377
photosynthetic electron transport (Krupa and Baszyński 1995; Burzyński and Kłobus 378
2004; Nagajyoti et al. 2010) as well as the activities of Calvin-Benson cycle enzymes 379
or the net assimilation of CO2 (Prasad and Strzałka 1999), Sgardelis et al. (1994)
380
showed that heavy metals did not influence PSII efficiency in Sonchus spp. and 381
Taraxacum spp. plants grown in urban environment. Trace metal pollution has been 382
shown to have no effect also on the chlorophyll content of some lichens species 383
(Chettri et al. 1998). However different findings are available in literature. For example, 384
Cu excess is demonstrated to cause in vitro photoinhibition of PSII, decrease of leaf 385
chlorophyll concentration and reduction of the thylakoid membrane network in 386
Phaseolus vulgaris L. (Pätsikkä et al. 2002). Furthermore, in two Zea mays L. cultivars, 387
Cu was found to decrease the quantum efficiency of PSII, ETR and qP, inducing also
388
a drop of leaf chlorophyll and carotenoids contents (Tanyolaç et al. 2007). 389
390
3.5 Antioxidant capacity and phenol content 391
The antioxidant capacity in shoot varied in a wide range between 7.3 and 305.3 µM 392
Trolox equivalent (TE) g-1 (mean of a 76.4), while in roots it ranged between 25.9 and
393
74.3 µM TE g-1 (Figure 4A). The antioxidant capacity of control was 132.6 and 40.5
394
µM TE g-1 for shoot and root portion, respectively. It is evident how the antioxidant
395
capacity was higher in shoot portion than in root independently to the site where plants 396
were grown (Figure 4A) and, with very few exceptions, it was similar in all monitored 397
sites. The exception are represented by sites 6, 19, 28 and 31, in which the shoot 398
portion of dandelion had a significantly high antioxidant capacity. On the other hand, 399
in these sites the shoot portion contained a high phenols content (Figure 4B), that 400
varied between 2.45 and 30.13 mg of gallic acid g-1. In the same sites, root phenol
401
amounts ranged between 2.80 and 6.97 mg of gallic acid g-1. Phenolic substances of
402
plants collected from the control site were 9.83 and 3.99 mg of gallic acid g-1 for shoot
403
and root, respectively. Phenolic compounds in shoot and root portion of control were 404
higher than those found in the same species by Zheng and Wang (2001), and were 405
13
similar to those found by Sengul et al. (2009) and by Hudec et al. (2007), which also 406
found a slightly higher antioxidant activity in the root than in the leaves. 407
The antioxidant capacity and the phenol content in shoot portion of dandelion 408
were positively correlated with Cu and Zn contents in soil (Table 5). The antioxidant 409
capacities of both parts of the plant were strongly correlated each other and with their 410
own phenolics content, as shown in Table 6. This because phenols are generally 411
recognized as one of the most active groups of antioxidants (Bors and Michel 2002; 412
Katalinic et al. 2006; Fraga et al. 2010). The antioxidant capacity of aerial and radical 413
part is related with the photochemical efficiency of PSII, while antioxidant capacity of 414
the aerial part showed a direct correlation also with the chlorophylls content. 415
416
4 Conclusions 417
The results reported in this work showed widespread very limited soil pollution of trace 418
metals in the urban sites. Soil reaction close to neutral and slightly alkaline limits the 419
mobility of the elements suggesting a scarce absorption by plants. On the other and, 420
results of metal accumulation in shoot and root tissues of dandelion confirm these 421
results, even though the ability of this species to exclude metals cannot be rejected. In 422
fact, BcF values (below the cut-value of 100) indicate a scarce assimilation of the 423
elements by dandelion, but also a poor translocation (TI values) from root to shoot. 424
Results showed also that the trace metals concentration into the tissues is below the 425
toxicity threshold. Zn that reached sometimes in root tissues the limits of toxicity 426
represents the only exception. However, no visible symptoms of damage were 427
recorded on leaf dandelion even if in plants in which Zn accumulated, the Fv/Fm ratio
428
decreased significantly indicating a negative effect of this element on photosynthetic 429
process. In addition, in these plants even an increase in phenols content and, 430
consequently, in antioxidant capacity, was found. This could indicate that although 431
dandelion accumulate Zn at level above toxicity (as confirmed also by the decrease in 432
Fv/Fm ratio) the high antioxidant capacity of leaves represents a response to the
433
oxidative load triggered by Zn. 434
Phenols play a key role as antioxidant moieties as evidenced by the their 435
increase in tissues of plants in which higher Zn content was found. 436
In conclusion, dandelion shows a high physiological performance that induces 437
in this species a high fitness in different environmental constrained conditions. This 438
14
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21 Legends of the Figures
644
Figure 1. Metal chelating capacity (MCC) of Taraxacum officinale shoot (white bar) 645
and root (grey bar) from plants grown in different monitored sites. Each value 646
represents the mean of three replicates. Different letters identify statistically different 647
means in accordance with Tuckey HSD test (P=0.05). 648
Figure 2. Chlorophyll fluorescence parameters (maximal photochemical quantum yield 649
of PSII, Fv/Fm; actual photochemical quantum yield of PSII, PSII; photochemical
650
quenching coefficient, qP; non-photochemical quenching coefficient, qN; and electron
651
transport rate, ETR) determined in leaves of Taraxacum officinale grown in monitored 652
sites. Each value represents the mean of three replicates. Different letters identify 653
statistically different means in accordance with Tuckey HSD test (P=0.05). 654
Figure 3. Pigment content in leaves of Taraxacum officinale collected in monitored 655
sites. Each value represents the mean of three replicates. Different letters identify 656
statistically different means in accordance with Tuckey HSD test (P=0.05). 657
Figure 4. Total antioxidant capacity (TEAC) and phenols content in shoot (white bar) 658
and root (grey bar) of Taraxacum officinale collected in monitored area. Each value 659
represents the mean of three replicates. Different letters identify statistically different 660
means in accordance with Tuckey HSD test (P=0.05). 661
22 663
Table 1. General features of urban soils of Pisa as compared to rural area of 664
San Rossore (Control). All parameters (n=3) are expressed as g 100 g-1 dry
665
soil. 666
Soil property Mean ±SD Minimum Maximum Control 667 Sand 68.1 10.8 40.1 85.2 72.9 668 Silt 16.8 6.4 7.9 30.8 16.4 669 Clay 15.1 5.5 6.9 31.6 10.7 670 pH 7.6 0.3 7.2 8.3 8.0 671 CaCO3 9.3 4.4 2.8 23.1 6.6 672 Organic C 3.3 1.2 1.3 7.6 0.9 673 674 675 676
23
Table 2. Heavy metals content (mg kg-1 soil) in urban soils of Pisa. Each
677
values represents the mean of three replicates. Different letters identify 678
different means in accordance with Tuckey HSD test (P=0.05). 679
Site Heavy metals 680 Cr Cu Mn Zn 681 0 39.8 n 32.2 pq 526 pq 51.0 r 682 1 115.0 c 124.9 b 668 ij 146.5 e 683 2 62.6 ef 69.3 hij 704 gh 100.9 jk 684 3 56.9 fgh 74.9 fg 765 ef 155.9 cde 685 4 48.5 lm 76.8 ef 746 f 133.7 f 686 5 52.4 hijklm 57.7 lmn 509 q 96.6 kl 687 6 49.4 klm 91.0 d 702 gh 96.7 kl 688 7 68.6 e 100.5 c 863 b 165.2 c 689 8 218 b 34.2 pq 632 lmn 90.9 klm 690 9 57.6 fghij 47.4 o 688 ghi 64.4 q 691 10 61.2 efg 61.2 klm 696 gh 117.3 hi 692 11 56.4 fghijk 77.7 ef 819 c 76.2 op 693 12 48.2 lm 74.1 fgh 606 no 84.3 mno 694 13 49.5 klm 70.0 ghi 687 ghi 160.0 cd 695 14 54.0ghijklm 62.5 kl 614 mno 110.2 ij 696 15 51.0 jklm 64.8 jk 658 jkl 131.2 fg 697 16 227.5 a 30.9 q 538 p 83.5 mno 698 17 56.8 fghijklm 56.2 mn 708 g 78.3 nop 699 18 51.7 ijklm 37.1 p 640 jkl 79.9 mnop 700 19 67.5 e 54.4 n 740 f 68.9 pq 701 20 58.9 fghi 54.1 n 704 gh 82.8 mno 702 21 59.2 fghi 62.6 kl 664 ijk 150.3 de 703 22 46.4 mn 57.2 mn 677 hij 205.3 b 704 23 61.5 efg 68.4 ij 804 cd 87.9 lmn 705
24 57.8 fghij 70.6 ghi 688 ghi 110.7 hij
706 25 94.4 d 80.8 e 1063 a 131.0 fg 707 26 55.9 fghijkl 63.0 k 700 gh 77.4 nop 708 27 52.9 hijklm 46.3 o 640 klm 90.3 klm 709 28 51.5 ijklm 44.7 o 589 o 61.7 qr 710 29 67.9 e 68.9 ij 798 cd 84.2 mno 711 30 57.1 fghijk 64.4 jk 782 de 121.5 gh 712 31 87.9 d 142.3 a 749 f 522.7 a 713 Average 71.2 67.4 705 121.5 714 715
24
Table 3. Heavy metals content (mg kg-1 dry weight) in shoot and root of Taraxacum officinale plants collected in monitored sites. Each
716
value represents the mean of three replicates. Different letters identify statistically different means in accordance with Tuckey HSD test 717
(P=0.05).- 718
Site
Cr Cu Mn Zn Cr Cu Mn Zn
0 2.7 de 16.7 def 23.3 def 116.7 abcdefg 8.7 bcde 14.7 def 70.0 bc 56.7 fg 1 2.0 e 15.3 def 25.3 def 83.3 cdefg 7.3 bcde 16.7 def 28.0 def 40.0 g 3 2.0 e 20.0 bcdef 23.3 def 113.3 abcdefg 4.0 cde 15.3 def 24.7 def 53.3 fg 4 10.0 bcd 20.0 bcdef 50.0 cdef 100.0 bcdefg 7.7 bcde 23.1 bcd 53.8 cd 53.8 fg 6 2.0 e 16.0 def 43.3 cdef 130.0 abcdef 6.0 cde 15.3 def 42.0 cdef 66.7 efg 7 5.5 cde 20.8 bcdef 40.9 cdef 173.1 ab 9.1 bcde 33.3 a 90.9 ab 151.2 abcde 8 5.7 cde 15.3 def 34.1 def 162.9 abc 10.2 bcd 22.3 bcdef 29.2 def 77.7 cdefg 13 4.6 cde 13.9 f 41.4 cdef 87.6 bcdefg 10.3 bcd 20.7 bcdef 72.4 bc 155.2 abcd 14 8.8 bcde 20.5 bcdef 49.5 cdef 123.9 abcdefg 9.0 bcde 14.7 def 19.7 ef 63,7 fg 15 5.4 cde 16.9 def 34.4 def 105.0 bcdefg 8.7 bcde 18.5 cdef 35.7 def 119.6 abcdefg 16 4.0 cde 16.0 def 30.7 def 83.3 cdefg 14.7 ab 17.3 def 27.3 def 80.0 cdefg 21 5.3 cde 15.7 def 73.2 bc 121.5 abcdefg 9.2 bcde 17.1 def 55.6 cd 78.5 cdefg 22 20.0 a 28.2 ab 107.9 a 196.8 a 9.4 bcde 22.6 bcde 23.7 def 65.2 efg 24 11.2 bc 16.3 def 48.9 cdef 123.7 abcdefg 14.5 ab 14.5 ef 29.1 def 111.8 abcdefg 27 3.3 cde 18.7 cdef 32.0 def 103.3 bcdefg 6.4 cde 20.1 bcdef 27.6 def 87.5 bcdefg 30 5.7 cde 19.6 cdef 41.2 cdef 63.1 fg 7.8 bcde 26.5 abc 51.9 cde 76.7 cdefg 31 2.0 e 17.3 def 19.3 f 106.7 bcdefg 4.0 cde 14.7 def 24.7 def 70.0 defg
Average 5.9 18.1 42.3 117.3 8.6 19.3 41.5 82.8
Shoot Root
25
Table 4. Indexes of heavy metals bioconcentration (BcF) or translocation (TI) in Taraxacum officinale plants collected in monitored sites. 720
Cr Cu Mn Zn
Site BcF TI BcF TI BcF TI BcF TI
Shoot root shoot root shoot root shoot root
0 6.7 bc 21.8 ab 0.3 b 51.7 a 45.5 bc 1.1 a 4.4 cdef 13.3 a 0.3 c 228.8 a 111.1 a 2.1 abc 1 1.7 c 6.4 cd 0.3 b 12.3 f 13.3 fg 0.9 a 3.8 def 4.2 c 0.9 bc 56.9 cd 27.3 bc 2.1 ab 3 3.4 c 6.7 cd 0.5 b 26.7 cdef 20.5 efg 1.3 a 3.1 ef 3.2 c 0.9 bc 72.7 cd 34.2 bc 2.1 ab 4 20.6 b 15.9 abc 1.3 ab 26.0 cdef 30.0 cdef 0.9 a 6.7 cde 7.2 bc 0.9 bc 74.8 cd 40.3 abc 1.9 abc 6 4.0 c 12.1 bcd 0.3 b 17.6 ef 16.8 efg 1.0 a 6.2 cdef 6.0 bc 1.0 bc 134.4 bc 68.9 abc 2.0 abc 7 8.0 bc 13.3 bcd 0.6 b 20.7 def 33.2 cde 0.6 a 4.7 cdef 10.5 ab 0.4 bc 104.8 bcd 91.7 ab 1.1 bc 8 2.6 c 4.7 d 0.6 b 44.7 ab 65.0 a 0.7 a 5.4 cdef 4.6 c 1.2 bc 179.3 ab 85.5 abc 2.1 abc 13 9.3 bc 20.9 ab 0.4 b 19.8 def 29.5 cdef 0.7 a 6.0 cdef 10.5 ab 0.6 bc 54.8 cd 97.0 ab 0.6 c 14 16.3 bc 16.7 abc 1.0 b 32.8 bcd 23.4 defg 1.4 a 7.8 bc 3.2 c 2.4 b 112.5 bc 57.8 abc 1.9 abc 15 10.6 bc 17.0 abc 0.6 b 26.1 cdef 28.6 cdefg 0.9 a 5.2 cdef 5.4 bc 1.0 bc 80.0 cd 91.2 ab 0.9 bc 16 1.8 c 6.4 cd 0.3 b 51.7 a 56.0 ab 0.9 a 5.7 cdef 5.1 bc 1.1 bc 99.8 bcd 95.8 ab 1.0 bc 21 9.5 bc 15.7 abc 0.6 b 25.1 def 27.4 cdefg 0.9 a 11.0 b 8.4 abc 1.3 bc 80.8 cd 52.2 abc 1.5 abc 22 43.1 a 20.3 ab 2.1 a 49.3 a 39.6 bcd 1.2 a 15.9 a 3.5 c 4.5 a 95.8 bcd 31.7 bc 3.0 a 24 19.4 b 25.2 a 0.8 b 23.1 def 20.6 efg 1.1 a 7.1 cd 4.2 c 1.7 bc 111.8 bc 101.0 ab 1.1 bc 27 6.3 bc 12.1 bcd 0.5 b 40.3 abc 43.5 bc 0.9 a 5.0 cdef 4.3 c 1.2 bc 114.4 bc 96.9 ab 1.2 bc 30 10.0 bc 13.8 bcd 0.7 b 30.4 bcde 41.2 bcd 0.7 a 5.3 cdef 6.6 bc 0.8 bc 52.0 cd 63.1 abc 0.8 bc 31 2.3 c 4.6 d 0.5 b 12.2 f 10.3 g 1.2 a 2.6 f 3.3 c 0.8 bc 20.4 d 13.4 c 1.5 abc
26
Average 10.6 13.2 0.7 28.7 31.2 1.0 6.3 5.6 1.3 90.3 65.5 1.5
27
Table 5. Correlation matrix among soil heavy metals content and heavy metals and 722
some physiological and biochemical parameters in shoot and root of Taraxacum 723
officinale collected in the monitored sites.**: P=0.01; *: P=0.05; ns.: P>0.05. 724 Cr Cu Mn Zn Fv/Fm ns ns ns * -0.545 qP ns ns ns ns qN ns ns ns ns PSII ns ns ns ns ETR ns ns ns ns Chl a ns ns ns ns Chl b ns ns ns ns Total Chl ns ns ns ns carotenoids ns ns ns ns TEACshoot ns ** ns * 0.676 0.513 TEACroot ns ns ns ns Phenolicsshoot ns ** ns * 0.637 0.504 Phenolicsroot ns ns ns ns MCCshoot ns ns ns ns MCCroot ns ns ns ns Crshoot ns ns ns ns Crroot ns * ns ns -0.544 Cushoot ns ns ns ns Curoot ns ns * ns 0.544 Mnshoot ns ns ns ns Mnroot ns ns * ns 0.585 Znshoot ns ns ns ns Znroot ns ns ns ns 725
28
Table 6. Correlation matrix among Taraxacum officinale features. The coefficient of correlation used to discriminate (30 d.f) within 726
were: 0.449 for P=0.01 and 0.349 for P=0.05. **: P=0.01; *: P=0.05; ns.: P>0.05. 727
Fv/Fm qP qN PSII ETR Chl a Chl b Total Chl carotenoids TEACshoot TEACroot Phenolshoot Phenolroot MCCshootMCCroot
Fv/Fm 1 -0.798 0.882 0.890 0.881 -0.227 -0.240 -0.260 0.025 0.444 0.579 0.467 0.487 -0.285 -0.089 qP ** 1 -0.779 0.903 0.898 0.051 0.039 0.046 0.031 0.016 -0.150 -0.029 -0.220 0.286 0.343 qN ** ** 1 -0.934 -0.912 0.054 0.025 0.052 0.152 0.123 0.257 0.135 0.258 -0.197 -0.154 PS II ** ** ** 1 0.983 -0.040 0.014 -0.026 -0.099 -0.058 -0.210 -0.085 -0.198 0.235 0.313 ETR ** ** ** ** 1 -0.052 0.019 -0.031 -0.132 -0.054 -0.216 -0.083 -0.212 0.211 0.271 Chl a ns ns ns ns ns 1 0.511 0.910 0.782 -0.448 -0.306 -0.406 -0.297 0.250 0.088 Chlb ns ns ns ns ns ** 1 0.821 0.057 -0.355 -0.324 -0.291 -0.199 0.250 -0.021 Total Chl ns ns ns ns ns ** ** 1 0.547 -0.467 -0.355 -0.407 -0.286 0.293 0.046 carotenoids ns ns ns ns ns ** ns ** 1 -0.009 0.081 -0.002 -0.110 0.191 0.310 TEAChoot * ns ns ns ns * * ** ns 1 0.795 0.965 0.435 -0.115 0.408 TEAChroot ** ns ns ns ns ns ns * ns ** 1 0.853 0.644 -0.126 0.337 Phenolshoot ** ns ns ns ns * ns * ns ** ** 1 0.515 -0.017 0.382 Phenolroot ** ns ns ns ns ns ns ns ns * ** ** 1 0.183 0.438 MCChoot ns ns ns ns ns ns ns ns ns ns ns ns ns 1 0.219 MCCroot ns ns ns ns ns ns ns ns ns * ns * * ns 1 728
